Biomedical subjects

T Sekiya

Publications and source records attributed to T Sekiya.

At least 37 records · Page 2Linked to original sources

PubMed2000

Detection of amplification of a chromosomal fragment at 6p21 including the cyclin D3 gene in a glioblastoma cell line by arbitrarily primed polymerase chain reaction.

DNA from 10 human glioma cell lines was analyzed by arbitrarily primed polymerase chain reaction. By fingerprinting of the DNA fragments obtained, the presence of fragment Qx with an abnormal signal was detected in one of the glioblastoma cell lines, CCF-STTG1. The nucleotide sequence of this fragment of 387 base pairs showed no homology with any known sequences. Southern-blot analysis using Qx as a probe revealed that the abnormal signal was caused by amplification of DNA by about 50-fold. By analysis of radiation hybrid panels, the fragment was shown to be derived from a chromosomal region on 6p21. The cyclin D3 (ccnd3) gene and an EST locus, H40682, both of which were located in this region, were amplified by about 50-fold in this cell line. Two other loci, R75654 and M78872, flanking the Qx, CCND3 and H40682 loci, were not amplified, suggesting that the size of the amplicon was less than 62 cR. Since over-expression of the ccnd3 gene, but not the H40682 locus, was detected in the cell line CCF-STTG1, the increased amounts of cyclin D3 caused by gene amplification could be involved in the development and/or progression of this glioblastoma.

Blotting, Northern↗

PubMed2000

An in vivo quantifiable model of cochlear neuronal degeneration induced by central process injury.

In the available in vivo experimental models for cochlear neuronal degeneration, the peripheral (hair cell side) process of the cochlear nerve has been injured in order to induce neuronal degeneration. However, there has been no dependable experimental model in which cochlear neuronal degeneration begins from the central (brain stem side) process. This lack of a central process injury model has probably been due to the experimental difficulties that had to be overcome in order to reproducibly and selectively injure the central process of the cochlear neurons while maintaining the patency of the internal auditory artery in small experimental animals such as rats. Using rats, we first developed a central process injury model in which the reduction of the spiral ganglion cells due to retrograde degeneration of cochlear neurons can be quantitatively evaluated. In our experimental model, the cochlear nerve was compressed and injured by a compression-recording (CR) electrode placed at the internal auditory meatus. First, the cochlear nerve was compressed until the compound action potentials of the cochlear nerve became flat, and then the CR electrode was advanced by various compression speeds (5, 10, or 200 micrometer/s) to reach the same depth (400 micrometer). In our model, therefore, the reduction of the spiral ganglion cells was caused compression speed dependently. This method made it possible to produce compression injury to the cochlear nerve without evidence of damage to the blood supply to the cochlea via the internal auditory artery. This model gives us the means to obtain knowledge that was previously impossible to derive from the peripheral process injury models.

Animals↗

PubMed2000

Construction of human-rodent hybrid cells containing single transferable fragments of human chromosome 10p.

The introduction of chromosome 10p into human glioblastoma or prostate cancer cells has been demonstrated to suppress their malignant phenotype, suggesting the presence of glioma or prostate tumor suppressor genes on 10p. As a resource for the fine mapping of these genes, a series of human-rodent hybrid cell lines containing single transferable fragments (STFs) of 10p were constructed. Normal chromosome 10 tagged with a neomycin-resistance gene on its short arm was fragmented by gamma-irradiation of 5-10krad, transferred into mouse L cells or Chinese hamster ovary cells by microcell-mediated chromosome transfer (MMCT), and then selected against G418. Thirty-three independent rodent-human hybrids carrying various-sized STFs were obtained. Polymerase chain reaction (PCR)-based genotyping revealed that these STFs contained the whole, or portions, of a 43-cM region on 10p14-pter and could be defined by 19 sequence-tagged-site (STS) markers. Using this panel of hybrids as donors for further MMCT, genes on the refined fragments could be transferred into other cells. This hybrid panel would therefore be a useful resource for the fine mapping of the genes on 10p14-pter to segments of about 2.4 cM by functional complementation.

Animals↗

PubMed2000

A comprehensive classification system of vestibular schwannomas.

Because traditional classifications of vestibular schwannomas (according to relative size) cannot comprehensively describe lesions that grow in different patterns after arising in regions as diverse as the cerebellopontine (CP) angle, the internal auditory canal, and the region lateral to the fundus of the internal auditory canal (labyrinth), we developed a new system to classify vestibular schwannomas, a system that describes the anatomical structures involved by the tumour, rather than size alone. The vestibular schwannoma is classified first by location and then by extent. Our system provides surgeons information helpful in choosing the surgical approach, in estimating the difficulty of tumour excision, and in determining whether hearing might be preserved. Our system also avoids confusion and misunderstanding in discussions of treatment results because it reflects the diverse biological characteristics of vestibular schwannomas.

Ear, Inner↗

PubMed2000

Cerebellopontine angle cisternal infusion of NGF, BDNF and NT-3: effects on cochlear neurons disconnected from central target, cochlear nucleus. An in vivo quantitative study.

Cochlear neurons need their synaptic contacts with both their peripheral (organ of Corti) and central (cochlear nucleus) targets for survival. We examined the in vivo effectiveness of the neurotrophins (NGF, BDNF and NT-3) on cochlear neuronal survival using our in vivo model, in which the central connection alone was selectively and quantitatively interrupted. The particular neurotrophins evaluated in the present study did not appear to have cochlear nerve rescue potential. However, the experimental model reported here can serve as a useful tool to investigate cochlear neuronal degeneration from the central side, which may lead to identification of effective mediators in the future.

Animals↗

PubMed2000

Transgenic rats carrying human c-Ha-ras proto-oncogenes are highly susceptible to N-methyl-N-nitrosourea mammary carcinogenesis.

A rat line carrying three copies of the human c-Ha-ras proto-oncogene, including its own promoter region, was established and designated Hras128. Expression of the transgene was detected in all organs examined from Hras128 rats by northern blot analysis. To examine its influence on susceptibility to N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis, female rats were treated with 50 mg/kg MNU i.v. at 50 days of age. All 22 Hras128 transgenic rats rapidly developed multiple and large mammary carcinomas within as little as 8 weeks after MNU treatment (14.1 tumors/rat, average diameter 16.4 mm). In contrast, 24 non-transgenic littermates developed no or only small tumors (0.46 tumors/rat, average diameter 7.4 mm) within this period. PCR-restriction fragment length polymorphism (RFLP) analysis and direct sequencing for the transduced human c-Ha-ras proto-oncogene indicated that 38 out of 44 tumors (86.4%) contained cells with mutations at codon 12 in exon 1. However, the signal densities of the mutated bands observed in the RFLP analyses revealed the presence of mixed populations of mutated and non-mutated cells in the tumors, the latter being in the majority. PCR-single strand conformation polymorphism analysis detected no mutations in codons 12 or 61 of the endogenous rat c-Ha-ras gene of Hras128 rat tumors. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene.

Animals↗

PubMed2000

Intraoperative electrophysiological monitoring of oculomotor nuclei and their intramedullary tracts during midbrain tumor surgery.

OBJECTIVE: During surgery for intrinsic midbrain lesions, we intraoperatively recorded evoked compound muscle action potentials (ECMAPs) from the extraocular muscles and evaluated how this type of intraoperative electrophysiological monitoring could minimize postoperative oculomotor nerve palsy (ptosis and/or diplopia). METHODS: The ECMAPs were recorded through a spring electrode applied to the extraocular muscle (Method 1, seven cases) or a needle electrode inserted into the superior intraorbital space (Method 2, five cases). The surgeon repeated electrical stimulations whenever tissue of unknown origin was encountered intraoperatively, and this information was used to safely guide surgical resection of the tumors. RESULTS: Using these monitoring techniques, the response-free areas were resected and the areas from which ECMAP responses were recorded were avoided. For all 12 patients, ECMAPs were successfully recorded from the extraocular muscles. Ten patients did not exhibit any postoperative deterioration of oculomotor nerve function. Two patients exhibited deterioration of oculomotor nerve function immediately after surgery, which resolved within 1 month. Equally robust ECMAPs could be recorded with Method 2, compared with Method 1. CONCLUSION: Intraoperative ECMAP recordings from the extraocular muscles precisely indicated the locations of the oculomotor nuclei and/or intramedullary oculomotor tracts. Although Method 2 is a more indirect method for recording ECMAPs than is Method 1, Method 2 was equally useful in recording ECMAPs, which seemed to be the summed potentials from the superior rectus muscle and the levator palpebrae superioris muscle. These monitoring techniques are valuable in guiding surgeons to avoid causing inadvertent harm to the oculomotor nuclei and tracts during midbrain surgery, particularly when the neuroanatomic features are distorted by the presence of tumor.

Adolescent↗

PubMed2000

Molecular analysis of the inhibition of monocyte chemoattractant protein-1 gene expression by estrogens and xenoestrogens in MCF-7 cells.

Xenoestrogens (XEs) are a diverse group of chemicals that mimic estrogenic actions and may have adverse effects on human health. The influence of these compounds on cytokine production or immune system function remains unclear. In this study we have examined the effects of 17beta-estradiol (E2) and XEs on chemoattractant cytokine (chemokine) production and analyzed the molecular mechanism. Monocyte chemoattractant protein-1 (MCP-1), also termed monocyte chemotactic and activating factor, is a member of the chemokine family and attracts mainly blood monocytes. Human mammary tumor cell line MCF-7 cells produce a large quantity of MCP-1 in response to interleukin-1alpha (IL-1alpha). Addition of E2 to MCF-7 cells inhibited MCP-1 production in a dose-dependent manner. XEs, bisphenol A, and NP also inhibited MCP-1 production, although the potency was 3-4 orders of magnitude lower than that of E2. E2, bisphenol A, and NP inhibited MCP-1 messenger RNA expression in MCF-7 cells. Two closely located nuclear factor-kappaB sites, A1 and A2, have been identified in the promoter of the human MCP-1 gene. A luciferase construct containing this enhancer region (pGLM-ENH) was activated by IL-1alpha, and a mutation at either the A1 or A2 site resulted in a loss of IL-1alpha responsiveness. Treatment with E2 or XEs decreased the IL-1alpha-inducible pGLM-ENH luciferase activity significantly. In an electrophoretic mobility shift assay and supershift analysis, we found that treatment with E2 or XEs diminished the IL-1alpha-induced complex formation with both A1 and A2 probes, which was identified immunochemically to consist of nuclear factor-kappaB, p50, and p65. The IL-1alpha-induced p50/c-Rel complex to the A2 probe was also, to a lesser extent, decreased by E2 or XE treatment. The effects of E2 and XEs on the expression of MCP-1 seem to be much more dramatic than the effects of these agents on the promoters used in the luciferase assay, suggesting the involvement of an additional site(s) of the promoter region of the MCP-1 gene or posttranscriptional regulation of MCP-1 gene expression by E2 and XEs. This work represents the first report describing possible regulation of immune system function by XEs through inhibiting chemokine production.

Blotting, Northern↗

PubMed2000

Effectiveness of preoperative administration of an N-methyl-D-aspartate antagonist to enhance cochlear neuron resistance to intraoperative traumatic stress: an experimental study.

OBJECT: Cochlear neurons are inevitably exposed to traumatic stress during surgical removal of an acoustic neuroma; that event is an important cause of postoperative cochlear neuronal degeneration, with subsequent loss of spiral ganglion cells (SGCs). The object of this study was to investigate whether preoperative pharmacological treatment can enhance the resistance of cochlear neurons to the traumatic stress of surgery. METHODS: Cochlear neuronal degeneration was induced in 17 rats by controlled compression of the cerebellopontine angle portion of the cochlear nerve. Dizocilpine maleate (MK-801; 10 mg/kg), an N-methyl-D-aspartate (NMDA) antagonist, was administered intraperitoneally to six of the 17 rats 30 minutes before compression occurred. Two weeks after compression, each rat was killed, and the numbers of SGCs in histological preparations of temporal bones were counted. CONCLUSIONS: Spiral ganglion cells were more numerous in rats administered dizocilpine maleate (p < 0.03) than in rats that did not receive treatment, indicating that receptor-mediated glutamate neurotoxicity may participate in the pathogenesis of trauma-induced cochlear neuron death and that administration of an NMDA antagonist before surgery may protect the nerve from injury leading to hearing loss.

Animals↗

PubMed2000

[Electrophysiological mapping of the trigeminal nerve root during microvascular decompression for trigeminal neuralgia].

A method for intraoperative electrophysiological mapping of the intracranial root of the trigeminal nerve was studied in five patients with trigeminal neuralgia. During surgery, the trigeminal nerve root was stimulated centrally with a bipolar electrode, and antidromic responses were recorded peripherally from three branches of the trigeminal nerve in the face. In all patients, the fibers of the individual subdivisions of the trigeminal nerve root were successfully localized based on the peripheral sites of antidromic response. This neural mapping was used during microvascular decompression in four patients and during a rhizotomy procedure in one patient. As a result of mapping, the fibers of the trigeminal division subserving the pain were clearly confirmed to be compressed by the artery in all four patients who were undergoing microvascular decompression. Likewise, the antidromic responses precisely identified the first division of the trigeminal nerve, which should be preserved to avoid postoperative corneal ulcers in patients undergoing rhizotomy. Based on these findings, it was concluded that this technique enables surgeons to precisely identify which fibers of the trigeminal nerve root should be decompressed or divided during surgery for trigeminal neuralgia.

Aged↗

PubMed2000

[Successful treatment of steroid-resistant bronchiolitis obliterans-organizing pneumonia with orally administered cyclosporin and pirfenidone].

A 27-year-old man with fever and dyspnea was admitted to our hospital. Chest computed tomography and a lung biopsy were performed, and bronchiolitis obliterans-organizing pneumonia (BOOP) was diagnosed. The patient was treated with corticosteroid, and a marked improvement was noted. However, when the dosage was tapered, BOOP recurred. Although the dosage was increased again, the corticosteroid alone was no longer effective against BOOP. While continuing with corticosteroid therapy, we also put the patient on a daily regimen of cyclosporin and pirfenidone, a recently developed anti-fibrotic agent. Both drugs were administered orally, and were so effective that we gradually decreased the dosage of corticosteroid. Several journals have reported that cyclosporin may be effective in the treatment of interstitial pneumonitis associated with collagen disease. We concluded that cyclosporin may also be useful in the treatment of refractory BOOP.

Administration, Oral↗

PubMed2000

Transgenic rats carrying copies of the human c-Ha-ras proto-oncogene exhibit enhanced susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine bladder carcinogenesis.

We have established a transgenic rat line carrying three copies of the human c-Ha-ras proto-oncogene with its own original promoter region, Jcl/SD-TgN(HrasGen)128Ncc (Hras128) rat. c-Ha-ras protein from expression of transduced and endogenous c-Ha-ras genes could be detected in the bladder epithelium of untreated transgenic rats. To examine their susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis, male transgenic and wild-type littermates were treated with 0.05% BBN in their drinking water for 10 weeks and then killed at week 20. The numbers and volumes of total macroscopic bladder tumors including both transitional cell papillomas and carcinomas (TCC) per rat were much greater in Hras128 rats than in their wild-type counterparts. The numbers of carcinomas per rat were also significantly greater in Hras128 rats. Two cases of TCC exhibiting invasion of the bladder muscle layer, which is extremely rare in the wild-type animals under the experimental conditions used, were also observed in Hras128 rats. The GGC-->GAC mutations at codon 12 of the transgene were observed in only two TCC out of 21 bladder tumors (9.5%), assessed by RFLP analysis and direct sequencing. SSCP analysis did not show any endogenous c-Ha-ras gene mutations. One of 25 tumors (4.0%) in wild-type rats had an endogenous c-Ha-ras gene mutation at codon 12 that was detected (GGA-->GAA) by single-strand conformation polymorphism and direct sequencing. These results indicate that the Hras128 rat is highly susceptible to BBN carcinogenesis and may be utilized as a rat model for analysis of bladder tumor development. The mutation findings indicate that the enhanced tumor development is not primarily due to mutations occurring in the transgene.

Animals↗

PubMed1999

Detection of DNA abnormalities by arbitrarily primed PCR fingerprinting: amplification of the MDM2 gene in a mediastinum fibrosarcoma.

Arbitrarily primed PCR (AP-PCR) fingerprinting method is easy and useful for analysis of genetic alterations in anonymous chromosomal regions. We applied this technology to analysis of DNA from human primary cancers and found amplification of a DNA fragment in a mediastinum fibrosarcoma. PCR-based analysis of radiation hybrid panels following cloning and nucleotide sequence determination of the fragment revealed that it was derived from a region of chromosome 12q13-q15. In this region, the MDM2 and IFNG genes were noted as known genes that could be involved in human carcinogenesis. Southern blot analysis of genomic DNA of the tumor revealed the amplification of the MDM2 gene together with the fragment locus, but not the IFNG gene. Our results demonstrated that detection of DNA alterations by AP-PCR fingerprinting without any previous knowledge of the genes and subsequent analysis of radiation hybrid panels could lead to easy identification of candidates for genes involved in carcinogenesis.

Base Sequence↗

PubMed1999

Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis.

We have constructed a library of DNA fragments heavily methylated in human adenocarcinomas of the lung to permit the comprehensive isolation of methylated CpG islands in cancer. Heavily methylated genomic DNA fragments from tumors of nine male patients were enriched using a methylated DNA binding column and used for construction of the library. From this library, DNA fragments having properties of CpG islands were isolated on the basis of their reduced rate of strand dissociation during denaturing gradient gel electrophoresis. Approximately 1,000 clones, corresponding to 0.3% of the library were analyzed, and nine DNA fragments were identified as being associated with CpG islands that were methylated in tumor DNA. One CpG island was methylated specifically in tumor DNA, whereas the remaining eight CpG islands were methylated both in normal and tumor DNA derived from the same patients. Our results suggest that the number of CpG islands methylated specifically in tumors is not large. The library, which contains DNA fragments from methylated CpG islands comprehensively, is expected to be valuable when elucidating epigenetic processes involved in carcinogenesis.

Adenocarcinoma↗

PubMed1999

A case of endolymphatic sac tumor with long-term survival.

A 72-year-old man developed left facial palsy at age 14 and left-sided hearing loss at age 20. At the age of 59, he presented with gait disturbance, and a large left cerebellopontine angle tumor was detected, which had markedly destroyed the pyramidal bone. The tumor was subtotally resected, but he required two more operations at the ages of 64 and 69 because of tumor regrowth. At the present time, recurrent tumor has destroyed the occipital bone and is invading the scalp. However, even though he has several cranial nerve palsies and cerebellar ataxia, he remains in stable condition and demonstrates long-term survival. The patient's surgical specimens revealed a papillary adenoma, which was recently thought to be of endolymphatic sac origin, although the origin of this kind of tumor, whether arising from the middle ear or from the endolymphatic sac, has not been established with certainty so far. In this paper, we provide further evidence that this tumor originates from the endolymphatic sac, based on anatomical, histopathological, and embryological evidence.

Adenoma↗

PubMed1999

Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia.

We investigated the alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia (T-ALL) and T-ALL cell lines by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Mutations of the p53 gene were found in three of 57 (5%) patients at diagnosis, one of 14 (7%) patients at relapse and in 12 of 18 (67%) cell lines. In these 12 cell lines, four had more than two mutations of the p53 gene. The p53 mutations were found in four of five cell lines whose original fresh leukemic cells were simultaneously examined original fresh leukemic cells. However, only one of the four fresh leukemic cells had the same mutation. All patients with p53 mutations in the course of disease died. Mutations of the p21 gene were not identified in 71 fresh samples and in 18 cell lines. N-RAS mutations were found in two of 57 (4%) fresh T-ALL patients at diagnosis, and four of 18 cell lines (22%), whereas no mutations were detected in any samples at relapse. Alterations of the p16 gene were found in 18 of 47 (38%) patients at diagnosis and in seven of 14 (50%) at relapse. These differences were not statistically significant. There were no differences in the frequency of alteration of the p16 and p15 genes between event-free patients and the remaining patients. Furthermore, we found the methylation of p16 gene in three of seven patients lacking homozygous deletions, suggesting higher frequency of p16 inactivation than previous reports in T-ALL. Interestingly, we found that one allele is inactivated by methylation and another allele had nonsense mutation in one cell line (KOPT-KI), resulting in loss of protein expression of p16. This type of p16 inactivation has not been so far reported in leukemia. We conclude that, (1) p53 mutations are infrequent at diagnosis but tend to be associated with poor clinical outcome; (2) RAS and p21 mutations may not be involved in the pathogenesis of T-ALL; (3) not only frequent alterations of p16 and p15 genes but also methylation of p16 gene are involved in initiating the leukemogenesis of T-ALLs, and (4) these 5 genes are independently involved in T-ALL.

Adolescent↗

PubMed1999

Effect of compression on the cochlear nerve: a short- and long-term electrophysiological and histological study.

The short- and long-term effects of static compression of the cochlear nerve were studied in dogs. The nerve was exposed in the cerebellopontine angle and a modified aneurysm clip was applied to reduce the diameter of the nerve trunk to 50%, 40%, 30% or 20% of normal (designated respectively as 50%, 60%, 70%, and 80% compression). Brainstem auditory evoked potentials (BAEPs) were monitored intraoperatively and post-operatively. The animals were sacrificed between 5 and 119 days after nerve compression and temporal bones were examined histologically. In the 50% compression group, all peaks except peak I disappeared immediately after nerve compression. After release of the clip, however, peak II and subsequent components recovered and prolonged interpeak latency (IPL) between peaks I and IV normalized within 7 days. In the 60% compression group, recovery was incomplete for as long as 49 days after compression. Significant histological changes were not always reflected in the electrophysiological recordings, as shown by the finding of multiple cavitations at the compressed portion of the cochlear nerve in cases in which conduction block of cochlear nerve impulses was reversible. In the 70% compression group, peak IV did not reappear for more than 1 week, and histological examination revealed severe damage to all cochlear nerve fibers except those from the apical turn, which lie in the center of the cochlear nerve trunk. Severe injury occurred to the cochlear nerve fibers that are situated more superficially in the nerve, which are tonotopically responsible for the perception of high-frequency sound and the generation of BAEPs. This means that the BAEP changes due to cochlear nerve compression would be detectable by BAEP monitoring, although changes in the apical region of the cochlea are not fully detectable by BAEP monitoring. In the 80% compression group, all peaks except peak I were lost permanently and the amplitude of peak I, which had been preserved in the acute phase, gradually decreased. Reversibility of impaired cochlear nerve impulse conduction was related to the severity of compression, and at some level of compression between 70% and 80% the nerve fibers generating BAEPs permanently lost the ability to conduct electrical impulses proximal to the site of compression. In the 70% and 80% compression groups, the amplitude of peak I gradually decreased over the first 30 days after compression and did not change significantly thereafter. Histologically, the branches of the internal auditory artery were resilient to compression, although they are easily avulsed due to stretch force. Furthermore, retrograde degeneration of cochlear neurons triggered by compression at the cisternal portion of the cochlear nerve was apparent. Such slowly progressive degeneration of nerve fibers may play a part in development of the delayed postoperative hearing disturbance.

Animals↗

PubMed1999

Alteration of mosaic methylation of the repeat unit of the human ribosomal RNA genes in lung cancer.

We have investigated the methylation status of the repeat unit of the human ribosomal RNA genes in lung cancer. Using a Southern blot analysis approach we have determined that the non-transcribed region of these genes was generally heavily methylated, while the transcribed region was not methylated in either tumor or normal DNA. Our study also revealed that, in one tumor, the boundary of mosaic methylation of the repeat unit was not distinct. In the same tumor, both the non-transcribed ribosomal spacer region and the L1 interspersed repeat sequences became partially demethylated. In tumor cells, the methylation status of DNA can be altered, but the methylation of subtelomeric repeats was found to be maintained. These results suggest that the mosaic methylation of the repeat unit is not necessarily maintained in tumor DNA, while subtelomeric repeats escape tumor-specific wave of demethylation.

Blotting, Southern↗